High throughput, multi-well assay systems are routinely used for target identification and lead optimization during drug development. More recently, high throughput systems have been proposed as a means to isolate high expressing cell lines necessary for commercial production of recombinant proteins. Edwards et al. (2004) Curr. Opin. Chem. Biol. 8:392-398 and U.S. Patent Publ. No. 2004/0033530A.
Fluorescence and bioluminescence labels typically are used in high throughput screening of biological samples. In techniques using fluorescence, cells of various tissue types are incubated in the presence of a fluorescent dye which is used as a label to identify a known target, marker or analyte. The signal emitted from the dye, after binding to the target, marker or analyte, is then detected using a camera or other detector. Disadvantages of fluorescent detection for biological assays include the small intensity of the fluorescence signal compared to the excitation intensity, the presence of background or nonspecific fluorescence, and interference from other fluorescence compounds than the label. For example, when measuring cells in a monolayer or microtiter plate, the cell layers are first illuminated with a light of a first wavelength and emission at a second wavelength is monitored as by a photodetector device. If any of the first wavelength leaks through the emission filter to the detector, or if any second wavelength is present in the excitation and reflects to the detector, they will contaminate the fluorescence signal emitted from the sample. Fluorescent label that is not completely washed or quenched and fluorescent compounds in the proximity of the cells can also affect the fluorescence signal from the label.
Bioluminescent labels avoid many of the drawbacks of fluorescence because they don't rely on excitation light and the sharp spectral filtering required to separate excitation and emission light. The major challenge of detecting bioluminescence is its inherently weaker intensity. Whereas with fluorescence, the signal can be increased by increasing the excitation intensity, dye concentration, and target density, bioluminescence is often limited by biological constraints on luminophore concentration.
Several multiplex systems are commercially available for use in high throughput screening but for various reasons, none of these interleave fluorescence and high-sensitivity luminescence imaging detection. For example, U.S. Pat. Nos. 6,057,163 and 6,985,225 describe sample reading devices for multiple sample analysis within a multi-well plate. Interference from adjacent samples is minimized by using a mask to isolate those wells or samples for detection while covering those which are not under analysis at that time. However, the process of masking and unmasking samples to be detected increases the amount of time necessary for calibration and analysis of a sample array. It also introduces moving mechanisms to automate the masking and unmasking of the samples in the device which increases the probability of device malfunction and error.
Moreover, none of these devices are suitable for multi-functional analysis of a complex biological or chemical sample using both fluorescent and luminescent labels integrated with fluidic transfers. Thus, a system that provides multi-functional fluidics and multimodal imaging of a complex sample which is amenable to automation and high throughput systems would be an advance over the prior art.